Cao Hang, Bissinger Rosi, Umbach Anja T, Gawaz Meinrad, Lang Florian
Department of Medicine III, Tuebingen, Germany.
Department of Physiology I, Eberhard-Karls-University, Tuebingen, Germany.
Cell Physiol Biochem. 2017;42(3):1252-1263. doi: 10.1159/000478954. Epub 2017 Jul 11.
BACKGROUND/AIMS: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus stimulates apoptosis of tumor cells and is thus therapeutically used for the treatment of diverse malignancies. On the other hand, temsirolimus has been shown to protect against apoptosis of hippocampal neurons. Similar to nucleated cells, blood platelets may enter suicidal death characterized by cell shrinkage and cell membrane scrambling. Platelet apoptosis is frequently preceded by Ca2+ entry, degranulation, integrin activation and stimulation of caspases. Those events could be triggered by collagen related peptide (CRP). The present study explored whether treatment of platelets with temsirolimus modifies platelet activation, caspase activity, platelet shrinkage, and phosphatidylserine abundance.
Platelets isolated from wild-type mice were exposed for 30 minutes to temsirolimus (40 µg/ml) without or with additional CRP (2 µg/ ml or 5 µg/ml) treatment. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fuorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding and relative platelet volume from forward scatter.
In the absence of CRP, the administration of temsirolimus (40 µg/ml) significantly decreased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity and aggregation. Exposure of platelets to CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, ROS, caspase activity and aggregation, effects significantly blunted in the presence of temsirolimus. CRP further decreased forward scatter, an effect again significantly blunted by temsirolimus.
Temsirolimus is a powerful inhibitor of platelet activation and suicidal platelet death.
背景/目的：雷帕霉素哺乳动物靶点（mTOR）抑制剂替西罗莫司可刺激肿瘤细胞凋亡，因此在治疗多种恶性肿瘤中具有治疗作用。另一方面，已证明替西罗莫司可保护海马神经元免于凋亡。与有核细胞类似，血小板可能进入以细胞收缩和细胞膜磷脂酰丝氨酸外翻为特征的自杀性死亡。血小板凋亡通常先有钙离子内流、脱颗粒、整合素激活和半胱天冬酶的刺激。这些事件可能由胶原相关肽（CRP）触发。本研究探讨了用替西罗莫司处理血小板是否会改变血小板激活、半胱天冬酶活性、血小板收缩和磷脂酰丝氨酸丰度。
将从野生型小鼠分离的血小板暴露于替西罗莫司（40μg/ml）30分钟，分别在无或有额外的CRP（2μg/ml或5μg/ml）处理的情况下。采用流式细胞术从Fluo-3荧光估计胞质钙离子活性（[Ca2+]i），从P-选择素丰度估计血小板脱颗粒，从αIIbβ3整合素丰度估计整合素激活，使用活性半胱天冬酶-3染色试剂盒测定半胱天冬酶活性，从膜联蛋白-V结合估计磷脂酰丝氨酸丰度，从前向散射估计相对血小板体积。
在无CRP的情况下，给予替西罗莫司（40μg/ml）显著降低[Ca2+]i，但未显著改变P-选择素丰度、活化的αIIbβ3整合素、膜联蛋白-V结合、细胞体积、半胱天冬酶活性和聚集。血小板暴露于CRP后，[Ca2+]i、P-选择素丰度、αIIbβ3整合素活性、膜联蛋白-V结合、活性氧、半胱天冬酶活性和聚集显著增加，在有替西罗莫司存在时这些效应显著减弱。CRP进一步降低前向散射，替西罗莫司再次显著减弱这一效应。
替西罗莫司是血小板激活和自杀性血小板死亡的强效抑制剂。
